PlGF is involved in important physiological and pathological processes, in particular angiogenesis. It plays an important part in tumor progression, kidney diseases, which are caused in particular by diabetes mellitus, in psoriasis, inflammatory diseases, in particular rheumatoid arthritis, in cardiovascular diseases and the like [Iyer, S.; Leonidas, D. D.; Swaminathan, G. J.; Maglione, D.; Battisti, M.; Tucci, M.; Persico, M. G.; Acharya, K. R. J Biol Chem 2001, 276, (15), 12153-61./Iyer, S.; Acharya, K. R. Trends Cardiovasc Med 2002, 12, (3), 128-34./Heeschen, C.; Dimmeler, S.; Fichtlscherer, S.; Hamm, C. W.; Berger, J.; Simoons, M. L.; Zeiher, A. M. JAMA 2004, 291, (4), 435-41./Yang, W.; Ahn, H.; Hinrichs, M.; Torry, R. J.; Torry, D. S. J Reprod Immunol 2003, 60, (1), 53-60.].
PlGF is mainly expressed in the placenta and belongs to the “cysteine-knot” protein family. PlGF occurs in different forms. Different forms of PlGF are (I) primary isoforms and (II) secondary isoforms. (III) In addition, a distinction can be made between free PlGF (fPlGF) and bound PlGF (gPlGF).
(I) Primary PlGF Isoforms
Primary PlGF isoforms are characterized by the primary sequence, i.e. the order of the amino acids in the protein. Alternative splicing and posttranslational modifications, such as glycosylations, phosphorylations, degradation (degradation products, fragments, etc.), acetylations etc., lead to different primary PlGF isoforms. To date, four different primary isoforms of human PlGF, PlGF-1 (PlGF-131), PlGF-2 (PlGF152), PlGF-3 (PlGF-203) and PlGF-4 have been described.
The sequence of the PlGF-1 precursor (Sequence number (SN) 1V) is as follows:
SN 1V(SEQ ID NO: 1)  1MPVMRLFPCF LQLLAGLALP AVPPQQWALS AGNGSSEVEV VPFQEVWGRS YCRALERLV D  61VVSEYPSEVE HMFSPSCVSL LRCTGCCGDE NLHCVPVETA NVTMQLLKIR SGDRPSYVEL 121TFSQHVRCEC RPLREKMKPE RCGDAVPRR
Secreted PlGF-1 does not as a rule possess the leader sequence of the PlGF-1 precursor (PlGF precursor) and thus begins at the N-terminus with alanine (A) (stated as A in the sequence of the PlGF-1 precursor, see above). This as a rule applies also to the other primary PlGF isoforms.
The sequence of the primary PlGF-1 isoform is thus as follows:
SN 1:(SEQ ID NO: 2)  1ALPAVPPQQW ALSAGNGSSE VEVVPFQEVW GRSYCRALER LVDVVSEYPS EVEHMFSPSC  61VSLLRCTGCC GDENLHCVPV ETANVTMQLL KIRSGDRPSY VELTFSQHVR CECRPLREKM 121KPERCGDAVP RR
In this primary sequence, possible sites for post-translational modifications, and hence also the presence of posttranslationally modified primary isoforms, can be discerned. For example, in general in vivo the posttranslationally modified primary PlGF isoform of PlGF-1 glycosylated at position 84 (asparagine, N) is present.
As the first N-terminal amino acid of the primary isoform PlGF-1, methionine (M) is often stated instead of alanine. This in general relates to recombinantly, for example in Escherichia coli (E. coli), expressed PlGF-1(rPlGF-1), and in particular to the human rPlGF-1 (rhPlGF-1). Here AUG, which codes for methionine, is used as the start codon. Such a PlGF expressed in E. coli has no posttranslational modifications, in particular also no glycosylations.
The sequence of the recombinant, human, primary PlGF-1 isoform is generally stated as follows:
SN 1RH:(SEQ ID NO: 3)  1MLPAVPPQQW ALSAGNGSSE VEVVPFQEVW GRSYCRALER LVDVVSEYPS EVEHMFSPSC  61VSLLRCTGCC GDENLHCVPV ETANVTMQLL KIRSGDRPSY VELTFSQHVR CECRPLREKM 121KPERCGDAVP RR
Through alternative splicing, the sequence RRRPKGRGKRRREKQRPTDCHL (SEQ ID NO: 64) is present in the PlGF-2 isoform instead of the arginine (R) 124. The sequence of the primary PlGF-2 isoform thus reads:
SN 2:(SEQ ID NO: 4)  1ALPAVPPQQW ALSAGNGSSE VEVVPFQEVW GRSYCRALER LVDVVSEYPS EVEHMFSPSC  61VSLLRCTGCC GDENLHCVPV ETANVTMQLL KIRSGDAPSY VELTFSQHVR CECRPLREKM 121KPERRRPKGR GKRRREKQRP TDCHLCGDAV PRR
An insert of 72 amino acids inserted by alternative splicing (HSPGRQSPDMPGDFRADAPSFLPPRRSLPMLFRMEWGCALTGSQS AVWPSSPVPEEIPRMHPGRNGKKQQRK (SEQ ID NO: 65)) leads to the sequence of the primary PlGF-3 isoform:
SN 3:(SEQ ID NO: 5)  1ALPAVPPQQW ALSAGNGSSE VEVVPFQEVW GRSYCRALER LVDVVSEYPS EVEHMFSPSC  61VSLLRCTGCC GDENLHCVPV ETANVTMQLL KIRSGDRPSY VELTFSQHVR CECRHSPGRQ 121SPDMPGDFRA DAPSFLPPRR SLPMLFRMEW GCALTGSQSA VWPSSPVPEE IPRMHPGRNG 181KKQQRKPLRE KMKPERCGDA VPRR
The primary PlGF-4 isoform contains sequences both of the PlGF-2 isoform (italic) and also of the PlGF-3 isoform (underlined):
SN 4:(SEQ ID NO: 6)  1ALPAVPPQQW ALSAGNGSSE VEVVPFQEVW GRSYCRALER LVDVVSEYPS EVEHMFSPSC  61VSLLRCTGCC GDENLHCVPV ETANVTMQLL KIRSGDRPSY VELTFSQHVR CECRHSPGRQ 121SPDMPGDFRA DAPSFLPPRR SLPMLFRMEW GCALTGSQSA VWPSSPVPEE IPRMHPGRNG 181KKQQRKPLRE KMKPERRRPK GRGKRRREKQ RPTDCHLCGD AVPRR(II) Secondary PlGF Isoforms
Secondary PlGF isoforms result from the combination of primary PlGF isoforms or other molecules, in particular molecules which are homologous to PlGF. The primary PlGF isoforms or other molecules are subunits of the secondary PlGF isoforms. In general, secondary PlGF isoforms consist of two subunits. Thus PlGF is as a rule present as a dimer, i.e. as a homodimer or a heterodimer. Homodimers consist of two identical primary PlGF isoforms (subunits) such as PlGF-1×PlGF-1, PlGF-2×PlGF-2, PlGF-3×PlGF-3 and PlGF-4×PlGF-4. Heterodimers consist of two different primary PlGF isoforms or of one primary PlGF isoform and one other molecule, in particular a PlGF homolog such as vascular endothelial growth factor (VEGF) and primary isoforms thereof. Possible examples of heterodimers are PlGF-1×PlGF-2, PlGF-3×PlGF-4, PlGF-1×VEGF, etc.
(III) Free PlGF (fPlGF) and Bound PlGF (gPlGF)
Since PlGF forms complexes with binding partners, the complexed or bound forms of PlGF must also be considered as well as the isoforms. In principle, the free primary, but in particular the free secondary PlGF isoforms (free PlGF, fPlGF), should be distinguished from the complexed or bound forms (bound PlGF, gPlGF). gPlGF is for example homodimeric PlGF-1 which is present in complexed form. These can be simple complexes, i.e. a PlGF-1 homodimer is bound to a receptor, for example the membrane-bound fms-like tyrosine kinase receptor-1 (mFlt-1). Other examples are complexes with the soluble Flt-1 (sFlt-1), with neurophilins (NP; in particular NP-1 and NP-2), with the kinase domain-containing receptor/fetal liver kinase receptor (KDR/Flk-1, VEGFR-2), with heparin sulfate proteoglycans (HSPG) and isoforms, homologs, fragments and degradation products thereof. Multilayer constituted complexes of several and sometimes different PlGF isoforms and several and sometimes different binding partners, in particular receptors, are also possible.
The function of PlGF is mediated, modulated or inhibited by binding to the membrane-bound or soluble fms-like tyrosine kinase receptor-1 (fms-like tyrosine kinase receptor-1 (Flt-1) or Vascular Endothelial Growth Factor (VEGF) receptor-1 (VEGFR-1)) and the kinase domain-containing receptor/fetal liver kinase receptor (KDR/Flk-1 or VEGFR-2). In addition to other possible functions of PlGF, the binding of PlGF to membrane-bound Flt-1 (mFlt-1) is especially important. This results in mFlt-1 transphosphorylation and thus activates signal transduction cascades [Iyer, S.; Acharya, K. R. Trends Cardiovasc Med 2002, 12, (3), 128-34.].
In contrast to this, it is presumed that the binding of PlGF to soluble Flt-1 (sFlt-1) serves to reduce the physiological activity of PlGF [Iyer, S.; Acharya, K. R. Trends Cardiovasc Med 2002, 12, (3), 128-34.]. Furthermore it is presumed that the PlGF isoform is involved. PlGF-2, which is possibly linked to the membrane, has a cationic insert of 21 amino acids at the carboxy terminal end. Through the binding of anionic, in particular polyanionic, substances such as heparin, heparin sulfate proteoglycans, etc., further functions can be mediated. The N-glycosylation of asparagine (Asn) 84 and the amino acid sequence which is present in PlGF-3 can also have similar effects. Moreover, it is presumed that the binding of PlGF and VEGF has yet another effect, since here the VEGF expression and thus its activity is negatively regulated [Iyer, S.; Acharya, K. R. Trends Cardiovasc Med 2002, 12, (3), 128-34.]. In summary, this means that the various forms of PlGF have different functions or exert different effects.
For the current detection methods and binding partners, in particular antibodies, which are at present used for analytical and diagnostic purposes, there is the problem that the different forms of PlGF are not, or not efficiently enough (not sufficiently specifically) distinguished. For example, the “Anti-human PlGF Antibody” from R&D Systems, Inc. does not exclusively recognize certain PlGF forms, in particular rhPlGF-1 homodimer but also the heterodimer of rhPlGF and VEGF and rhPlGF-2 (R&D Systems Catalog Number: AF-264-PB or DPG00 product descriptions).
Furthermore, fPlGF or gPlGF are not exclusively detected, i.e. with the existing antibodies, a distinction between fPlGF and gPlGF is not or not sufficiently efficiently made. In particular, the specific detection of fPlGF is inadequate. This is demonstrated by the fact that rhFlt-1 in the form of rhFlt-1/Fc has an effect on the determination of PlGF (R&D Systems Catalog Number: DPG00). This non-specificity is confirmed in the literature [Maynard, S. E.; Min, J. Y.; Merchan, J.; Lim, K. H.; Li, J.; Mondal, S.; Libermann, T. A.; Morgan, J. P.; Sellke, F. W.; Stillman, I. E.; Epstein, F. H.; Sukhatme, V. P.; Karumanchi, S. A. J Clin Invest 2003, 111, (5), 649-58.]. Maynard et al. show that the relevant R&D Systems ELISA (R&D Systems Catalog Number: AF-264-PB or DPG00) does show a certain specificity for fPlGF, however the studies performed show that this specificity is low. In the determination of 0.5 ng/mL rhPlGF-1, a signal reduction of only about 12% is to be seen in the presence of 0.5 ng/mL sFlt-1. Even with a 10-fold excess of sFlt-1 (5 ng/mL), there was only a signal reduction by the factor of 2. A more pronounced signal reduction would occur with higher specificity of the antibodies used towards fPlGF.